Low Light CMOS Contact Imager with an Integrated Poly-Acrylic Emission Filter for Fluorescence Detection

This study presents the fabrication of a low cost poly-acrylic acid (PAA) based emission filter integrated with a low light CMOS contact imager for fluorescence detection. The process involves the use of PAA as an adhesive for the emission filter. The poly-acrylic solution was chosen due its optical transparent properties, adhesive properties, miscibility with polar protic solvents and most importantly its bio-compatibility with a biological environment. The emission filter, also known as an absorption filter, involves dissolving an absorbing specimen in a polar protic solvent and mixing it with the PAA to uniformly bond the absorbing specimen and harden the filter. The PAA is optically transparent in solid form and therefore does not contribute to the absorbance of light in the visible spectrum. Many combinations of absorbing specimen and polar protic solvents can be derived, yielding different filter characteristics in different parts of the spectrum. We report a specific combination as a first example of implementation of our technology. The filter reported has excitation in the green spectrum and emission in the red spectrum, utilizing the increased quantum efficiency of the photo sensitive sensor array. The thickness of the filter (20 μm) was chosen by calculating the desired SNR using Beer-Lambert’s law for liquids, Quantum Yield of the fluorophore and the Quantum Efficiency of the sensor array. The filters promising characteristics make it suitable for low light fluorescence detection. The filter was integrated with a fully functional low noise, low light CMOS contact imager and experimental results using fluorescence polystyrene micro-spheres are presented

CMOS SINGLE-PHOTON AVALANCHE DIODES AND MICROMACHINED OPTICAL FILTERS FOR INTEGRATED FLUORESCENCE SENSING

This dissertation presents a body of work that addresses the two most pressing challenges in the field of integrated fluorescence sensing, namely, the design of integrated optical sensors and the fabrication of high-rejection micro-scale optical filters. Two novel enabling technologies were introduced. They are: the perimeter-gated single-photon avalanche diode (PGSPAD), for on-chip photon counting, and the benzotriazole (BTA)-doped thin-film polymer filter, for on-chip ultraviolet light rejection. Experimental results revealed that the PGSPAD front-end, fabricated in a 0.5 μm standard mixed-signal CMOS process, had the capability of counting photons in the MHz regime. In addition, it was found that a perimeter gate, a structural feature used to suppress edge breakdown in the diode, also maximized the signal-to-noise-ratio in the high-count rate regime whereas it maximized sensitivity at low count rates. On the other hand, BTA-doped filters were demonstrated utilizing three commonly used polymers as hosts. The filters were patternable, utilizing the same procedures traditionally used to pattern the undoped polymer hosts, a key advantage for integration into microsystems. Filter performance was analyzed using a set of metrics developed for optoelectronic characterization of integrated fluorescence sensors; high rejection levels (nearing -40 dB) of UV light were observed in films of only 5 μm in thickness. Ultimately, BTA-doped filters were integrated into a portable sensor, and their use was demonstrated in two types of bioassays

Towards Integrated Fluorescence Sensing

This thesis is an account of ongoing efforts in the Integrated Biomorphic Information Systems Laboratory and the Laboratory for MicroTechnologies towards the implementation of integrated microfabricated biosensing platforms with on-chip fluorescence detection capability. The first chapter is a published, exhaustive, and critical review of state-of-the-art microfluorometers, and it offers a set of performance metrics for evaluating sensors of different architectures. The second chapter consists of material from two journal papers, currently in preparation, in which the development of a polymeric optical filter material for UV fluorescence spectroscopy is presented and its integration with a CMOS active pixel sensor (APS) discussed. The third chapter, which is also an archival publication, presents initial efforts towards achieving high-sensitivity CMOS photodetectors for photon counting-based fluorescence assays in integrated platforms

NASA Tech Briefs, December 2007

Topics include: Ka-Band TWT High-Efficiency Power Combiner for High-Rate Data Transmission; Reusable, Extensible High-Level Data-Distribution Concept; Processing Satellite Imagery To Detect Waste Tire Piles; Monitoring by Use of Clusters of Sensor-Data Vectors; Circuit and Method for Communication Over DC Power Line; Switched Band-Pass Filters for Adaptive Transceivers; Noncoherent DTTLs for Symbol Synchronization; High-Voltage Power Supply With Fast Rise and Fall Times; Waveguide Calibrator for Multi-Element Probe Calibration; Four-Way Ka-Band Power Combiner; Loss-of-Control-Inhibitor Systems for Aircraft; Improved Underwater Excitation-Emission Matrix Fluorometer; Metrology Camera System Using Two-Color Interferometry; Design and Fabrication of High-Efficiency CMOS/CCD Imagers; Foam Core Shielding for Spacecraft CHEM-Based Self-Deploying Planetary Storage Tanks Sequestration of Single-Walled Carbon Nanotubes in a Polymer PPC750 Performance Monitor Application-Program-Installer Builder Using Visual Odometry to Estimate Position and Attitude Design and Data Management System Simple, Script-Based Science Processing Archive Automated Rocket Propulsion Test Management Online Remote Sensing Interface Fusing Image Data for Calculating Position of an Object Implementation of a Point Algorithm for Real-Time Convex Optimization Handling Input and Output for COAMPS Modeling and Grid Generation of Iced Airfoils Automated Identification of Nucleotide Sequences Balloon Design Software Rocket Science 101 Interactive Educational Program Creep Forming of Carbon-Reinforced Ceramic-Matrix Composites Dog-Bone Horns for Piezoelectric Ultrasonic/Sonic Actuators Benchtop Detection of Proteins Recombinant Collagenlike Proteins Remote Sensing of Parasitic Nematodes in Plants Direct Coupling From WGM Resonator Disks to Photodetectors Using Digital Radiography To Image Liquid Nitrogen in Voids Multiple-Parameter, Low-False-Alarm Fire-Detection Systems Mosaic-Detector-Based Fluorescence Spectral Imager Plasmoid Thruster for High Specific-Impulse Propulsion Analysis Method for Quantifying Vehicle Design Goals Improved Tracking of Targets by Cameras on a Mars Rover Sample Caching Subsystem Multistage Passive Cooler for Spaceborne Instruments GVIPS Models and Software Stowable Energy-Absorbing Rocker-Bogie Suspension

Diagnostic Biosensors for Detection of Blood-Derived Biomarkers

Standard diagnostic tools used from patient samples, specifically from blood draws, require specialized equipment, personnel, and facilities. Conventional techniques can often be very laborious and time consuming due to required sample preparation. The evident delay from sample collection to a patient’s result immensely impacts their outcome. The aims of this research are to design diagnostic biosensors that decrease time-to-results, minimize reagent and sample handling, and incorporate automated simple optical transduction and user interfaces for the detection of blood-derived biomarkers. Specifically, four biosensing detection mechanisms performed on 3 different point-of-care platforms will be discussed. First is a static loop-mediated isothermal amplification (LAMP) of nucleic acid aqueous droplet on a silicone chip platform immersed in mineral oil. The target-of-interest is a nucleic acid sequence as a biomarker for antibiotic resistant bacteria. The biosensing technique used related changes in interfacial tension (IFT) at the water-oil interface by measuring the change in contact angle (geometrical-effects) over time. Initially the system was characterized as a linear response in relation to concentration of bacteria in a buffer system down to the limit of detection (LOD) of 100 CFU per uL. Subsequently, with the addition of bacterial infected blood sample models, the system became a binary assay (i.e. yes or no) as low as 10 CFU per uL within 10 min of reaction. Secondly, a two-layered, paper microfluidic chip was utilized to quantify cancer cells from a buffy coat sample matrix by two detection mechanisms: 1) on-chip particle enumeration via smartphone microscope and 2) capillary flow dynamics via smartphone video processing. The assay resulted in a LOD as low as 1 cell per uL for the on-chip imaging aspect of platform and 0.1 cell per uL for the capillary flow analysis within 13 to 22s post application of blood sample. Lastly, the same concepts previously described in the first platform utilizes changes in IFT due to amplicon presence in an aqueous solution immersed in mineral oil. An emulsion LAMP platform was investigated to determine the relation between angle-dependent light scatter intensity (based off Mie scatter theory) and nucleic acid amplification progression. The phenomenon attributing to changes in light scatter intensities is due to the interfacial changes occurring in the emulsion droplets, where amplicon amount increases the IFT decreases, resulting in smaller diameter emulsions. Changes in light scatter intensity within 3 min of the reaction shows statistical difference in comparison to no target control (NTC) for 10^3 CFU per uL of bacteria dosed into aqueous sample. These four detection mechanisms and three platforms offer but a few alternatives as biosensing methods for blood-derived diagnostic biosensors

All-in-one microsystem for long-term cell culturing and real-time chip-level lensless microscopy

The study is to concept, develop and evaluate an all-in-one microsystem with combined long-term animal cell culturing and real-time chip-level lensless microscopy functions suitable for applications in cell biology studies, and at the point-of-use. The microsystem consists of a 5 megapixel CMOS image sensor, a disposable microchip for cell culture, heating elements and LED illumination. The overall size is only 40 mm x 40 mm x 50 mm. The disposable microchip for cell culture is composed of a polymer microfluidic interface and a silicon micro-cavity chip with a 1 µm thick 1 mm x 1 mm transparent Si3N4 bottom membrane, which is directly placed onto the image sensor surface. Under the collimated LED illumination, the optical resolution of the lensless imaging module is only dependent on the digital resolution of the image sensor, which amounts to 3.5 µm (double pixel pitches). The imaging quality is proven comparable to a 4x optical microscope without image computation or processing. Both the morphologies of different cell cultures (L929, A549 and T47D) and the single cells with colorimetric staining can be clearly visualized in real time. With the additional deposition of an interference filter on the image sensor surface, fluorescence spreading cells in culture are observed on the chip under a common blue LED illumination. The temperature for the incubating module is controlled at 37±0.2°C in the room environment. Mammalian cells (L929 and A549) are cultured with conventional culture medium and monitored under the time-lapse lensless microscopy by the all-in-one microsystem up to 5 days outside a laboratory incubator. Very fast operational processes, such as cell loading, passaging and staining, have been readily carried out and monitored in real-time by the platform. Besides cell cultures in monolayer, the formation of 3D clusters of L929 cells has also been demonstrated and recorded under time-lapse lensless microscopy by using the all-in-one microsystem.Das Ziel der vorliegenden Dissertation ist die Konzeption, Entwicklung und Evaluation eines All-in-One-Mikrosystems mit der Kombination aus Langzeit Kultivierung von Tierzellen und Echtzeit Linsenloser Mikroskopie Funktionen auf Chip Level, die für Anwendungen in zellbiologischen Studien sowie für Point-of-use geeignet sind. Das Mikrosystem besteht aus einem 5 Megapixel CMOS-Bildsensor, einem Einweg-Mikrochip für die Zellkultur, Heizelementen sowie einer LED-Beleuchtung Die Gesamtgröße beträgt nur 40 mm x 40 mm x 50 mm. Der Einweg-Mikrochip für die Zellkultur besteht aus einer polymeren, mikrofluidischen Grenzfläche und einem Silizium-Mikrohohlraum-Chip mit einer 1 µm dicken und 1 mm x 1 mm transparenten Si3N4-Bodenmembran, die direkt auf die Bildsensoroberfläche aufgesetzt wird. Unter der kollimierten LED-Beleuchtung ist die optische Auflösung des linsenlosen Abbildungsmoduls nur von der digitalen Auflösung des Bildsensors abhängig, was 3,5 µm beträgt (Doppelpixelabstände). Die Bildqualität ist vergleichbar mit einem 4x optischen Mikroskop ohne Bildberechnung oder Verarbeitung. Sowohl die Morphologien verschiedener Zellkulturen (L929, A549 und T47D) als auch die einzelnen Zellen mit farbmetrischer Färbung können in Echtzeit deutlich sichtbar gemacht werden. Mit der zusätzlichen Abscheidung eines Interferenzfilters auf der Bildsensoroberfläche werden fluoreszenzverteilende Zellen in Kultur auf dem Chip unter einer gemeinsamen blauen LED-Beleuchtung beobachtet. Die Temperatur für das Inkubationsmodul wird bei 37 ± 0,2 ° C in der Raumumgebung festgelegt. Säugetierzellen (L929 und A549) werden mit herkömmlichem Kulturmedium kultiviert und unter der Zeitrafferlinsenmikroskopie durch das All-in-One-Mikrosystem bis zu 5 Tage außerhalb eines Laborinkubators überwacht. Sehr schnelle operative Prozesse wie z. B. Zellbeladung, Durchfluss und Färbung wurden in Echtzeit durch die Plattform durchgeführt und überwacht. Neben den Zellkulturen in der Monoschicht wurde auch die Bildung von 3D-Clustern von L929-Zellen in Zeitraffer bei lichtempfindlicher Mikroskopie unter Verwendung des All-in-One-Mikrosystems nachgewiesen und aufgezeichnet

PRINCIPLES FOR NEW OPTICAL TECHNIQUES IN MEDICAL DIAGNOSTICS FOR mHEALTH APPLICATIONS

Medical diagnostics is a critical element of effective medical treatment. However, many modern and emerging diagnostic technologies are not affordable or compatible with the needs and conditions found in low-income and middle-income countries and regions. Resource-poor areas require low-cost, robust, easy-to-use, and portable diagnostics devices compatible with telemedicine (i.e. mHealth) that can be adapted to meet diverse medical needs. Many suitable devices will need to be based on optical technologies, which are used for many types of biological analyses. This dissertation describes the fabrication and detection principles for several low-cost optical technologies for mHealth applications including: (1) a webcam based multi-wavelength fluorescence plate reader, (2) a lens-free optical detector used for the detection of Botulinum A neurotoxin activity, (3) a low cost micro-array reader that allows the performance of typical fluorescence based assays demonstrated for the detection of the toxin staphylococcal enterotoxin (SEB), and (4) a wide-field flow cytometer for high throughput detection of fluorescently labeled rare cells. This dissertation discusses how these technologies can be harnessed using readily available consumer electronics components such as webcams, cell phones, CCD cameras, LEDs, and laser diodes. There are challenges in developing devices with sufficient sensitivity and specificity, and approaches are presented to overcoming these challenges to create optical detectors that can serve as low cost medical diagnostics in resource-poor settings for mHealth

Inverted Fluorescence Microscope

The Inverted Fluorescence Microscope senior project team at Cal Poly, San Luis Obispo designed, assembled, and tested a proof-of-concept inverted fluorescence microscope for the university’s Microfabrication Laboratory. Administrators of the laboratory wished to use fluorescence for research and experiments involving cell growth and flow visualization on the micro-scale, and did not have the budget to purchase one of the costly commercially available options. The scope of this design challenge was to produce a low-cost inverted fluorescence microscope employing available optical components and additional readily sourced parts to expand the use of fluorescence microscopy accessible to undergraduate students in the Microfabrication Laboratory. This document is an account of the final microscope design as well as the engineering design process, project management procedures, and timeline followed to produce a working design verification prototype. The final product successfully resolved images of microfluidic devices in brightfield mode with automated maneuverability in the X-Y plane. It is equipped with fluorescence capabilities, and will serve as a valuable, low-cost research tool and platform for future student projects

Microfluidic paper-based analytical devices with instrument-free detection and miniaturized portable detectors

icrofluidic paper-based analytical devices (mu PADs) have attracted much attention over the past decade because they offer clinicians the ability to deliver point-of-care testing and onsite analysis. Many of the advantages of mu PADs, however, are limited to work in a laboratory setting due to the difficulties of processing data when using electronic devices in the field. This review focuses on the use of mu PADs that have the potential to work without batteries or with only small and portable devices such as smartphones, timers, or miniaturized detectors. The mu PADs that can be operated without batteries are, in general, those that allow the visual judgment of analyte concentrations via readouts that are measured in time, distance, count, or text. Conversely, a smartphone works as a camera to permit the capture and processing of an image that digitizes the color intensity produced by the reaction of an analyte with a colorimetric reagent. Miniaturized detectors for electrochemical, fluorometric, chemiluminescence, and electrochemiluminescence methods are also discussed, although some of them require the use of a laptop computer for operation and data processing